Ornithine decarboxylase: An oncogene?

Ornithine decarboxylase: An oncogene?

SELECTED SUMMARIES October 1993 the light tioned, of the muscle demonstration cells with strong strip and pathophysiological that VZV a coord...

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SELECTED SUMMARIES

October 1993

the light tioned,

of the muscle demonstration

cells with strong

strip

and pathophysiological

that

VZV

a coordinating

support

is confined

and inhibitory

studies to those

function

men-

would

provide

for our hypothesis.

The localization of achalasia to the esophagus suaests a local lesion rather than one in the central nervous system or the vagus nerve.

In our view, ganglion

the consequence

cell depletion

of esophageal

to sorne degree

in vigorous

is unlikely

distention, achalasia

because

before

to be simply

it can be shown

dilatation

has devel-

oped. We join Dr. Schulze-Delrieu cal role of VZV in achalasia confirm

or refute

myenteric ganglion

plexus

in hoping

and to localize which,

in specific

is a more

PROF.

to

in the esophageal

its presence

of course,

studies types

daunting

MICHAEL

of

ATKINSON

of Helsinki,

sense construct had a reduced growth rate. ODC transfected contrast

to the antisense

ODC

transfectants

Expression of human ODC by the pHPApr1 neo vector or the PMAMneo

vector

(dexamethasone

inducible)

cells with a lower efficiency

led to

of transfection

than the pLTRpoly/ODC

construct, reflecting different po-

tencies of the promoters.

When these vectors had the ODC

cDNA in the reverse direction,

no morphological

Additionally,

Southern

transfor-

blot analysis

indicated that human ODC was stably integrated into genoCells with the pLTRpoly/ODC

Helsinki,

Finland).

of Or-

PMAMneo

construct

ity, respectively.

ODC

activity

remained

versely, in the normal cells, ODC

26).

In this report from Auvinen et al., the authors studied the role of ornithine

decarboxylase

and oncogenesis.

A key regulator of polyamine

(ODC)

in transformation synthesis,

ODC is an enzyme whose expression is transiently increased and is constitutively However,

acti-

it is unclear

and

constructs had about 60% and 30% of that activ-

tion. Nature 1992;360:355-358

(November

had very high

ODC activity, whereas the pHPAprlneo

stable

vated during cell transformation.

and the un-

transfected cells.

nithine decarboxylase activity is critical for cell transforma-

upon growth factor stimulation

more rapidly than

and the cells with ODC anti-

cells could grow in low serum (0.5% fetal calf serum) in

constitutive

Auvinen M, Paasinen A, Andersson LC, et al. (Department University

controls,

mic DNA derived from the transfectants.

ORNITHINE DECARBOXYLASE: AN ONCOGENE? Pathology,

the untransfected

mation was evident.

task.

of antisense ODC. Cells

by ODC cDNA proliferated

transformed

etiologi-

by further

of VZV DNA

the presence

cells there,

that the possible

will be elucidated

were present with the transfection transformed

ganglion

1255

transfectants,

even

after

serum

high in all the starvation.

Con-

activity was negligible

after serum starvation. The authors then investigated whether the augmentation in ODC production

and its transforming

activity was asso-

ciated with changes

in tyrosine

upon the observation

that alpha-difluoromethyl

(DFMO),

phosphorylation,

based

ornithine

an inhibitor of ODC, causes a decrease in protein

whether ODC is a direct mediator or an innocent bystander

tyrosine phosphorylation.

Western blot analysis revealed a

of cell transformation.

protein of 130 kilodaltons,

which undergoes increased tyro-

tion,

the authors

To address that fundamental

inserted

human

ODC

ques-

complementary

sine phosphorylation

in the ODC transfectants

compared

DNA (cDNA) into three expression vectors with promoters

with the parental cells. The identity of this protein is un-

of different

clear. The authors further observed that blocking ODC by

cells.

strength

Concurrently,

marker (pSV2neo) resistant

and transfected control

plasmids

were transfected.

cells were isolated

growth and transforming

them into NIH3T3 and

a selectable

Thereafter,

neomycin-

after two weeks,

and their

properties were investigated.

The three expression vectors in which the human ODC cDNA was inserted were pLTRpoly

(contains long terminal

repeat [LTR] of Moloney murine leukemia virus as the promoter and the SV40 splicing and polyadenylation

signals),

pMAM neo (Rous sarcoma virus LTR enhancer

linked to

the dexamethasone-inducible

mouse mammary tumor virus

LTR promoter and SV40 splicing and polyadenylation plus the neomycin-resistance tains the human quence-l

P-actin

with enhancer

gene), and pHPAprlneo promoter

sites (con-

plus intervening

se-

like activity and the SV40 poly-

adenylation signal). All of these expression vectors were also assembled with human ODC cDNA in the antisense direction to serve as controls during transfections. When pLTRpoly/ODC was transfected into NIH3T3 cells, there was complete morphological transformation. In particular, the cells had no contact inhibition, ple foci

in monolayer

their actin filaments, colonies

formed multi-

cultures, displayed disintegration

of

and had acquired the ability to form

in soft agar. Conversely,

none of these features

DFMO inhibits transformation of rat fibroblasts by v-m. The authors conclude that ODC is a requisite for transformation,

and the degree of transformation

is tied to the

expression of ODC as indicated by the use of different expression vectors. Anything

but an innocent

bystander, the

authors suggest that ODC plays a significant

role in onco-

genesis.

Comment. mine

The

are normal

polyamines

in cell proliferation cells

depleted

of polyamines with

1988;48:759-774).

Based

lieved

that polyamines

charge

of DNA

amines

stabilize

RNA.

It is likely

which

polyamines

The

pathway

that

resume

on their

chemical

DNA

by phosphate RNA,

these

role

experiments:

polyamine-depleted growth

(Cancer

structures,

by neutralizing groups.

messenger

the negative

RNA,

and

provided

and protein

RNA,

synthesis to putrescine,

step in polyamine

from putrescine

in cells

decarboxylase.

by

synthesis.

is in large

part

catalyzes

is the first and

synthesis.

is catalyzed

polytransfer

the means

ODC

which

Res

it is be-

Additionally,

interactions

ornithine

of ornithine

sper-

DNA,

for polyamine

the rate-limiting

tion of spermidine

growing;

stimulate

by the enzyme

the conversion usually

stop

ribosomal

and

play a significant

by the following

polyamines stabilize

provided

spermidine,

of cells. They

as evidenced

cells supplemented

regulated

putrescine,

components

The forma-

by spermidine

syn-

1256

SELECTED

SUMMARIES

thase,

and in turn,

spermidine

thase.

The

derived

aminopropyl

from

of polyamine

specific

inhibitors,

(DFMO),

a highly

specific

The

mammalian

oncogene

the enzyme

often

amplification

have

been

of about

rat, mouse)

of ODC

and a predicted

(Trends

stimulation. increase

in ODC

synthesis

Another

ODC

malignancies Investigations

colon

carcinomas

1275-1281),

(Ann

Med

1984;311:80-83).

vated

in normal

administration

Surg

stimuli,

enhanced

fected

syn-

protein

1986;204:89-93;

of gastrin

(Am J Physiol

a marker

Thus,

of cellular

proteins

transformation

(Cancer

A key question

regulators

are indirect

cDNA

into

moters

of various

a mammalian

promoters

of

strengths.

can be transfected

cells such that the cDNA and protein

tyrosine

kinase

yet to be identiin the colon. is whether

transformation

is addressed

strategy

is the region elements

or introduced

into cultured

is transcribed

into RNA

parental,

or untransfected,

levels

in the transfected

in

in markedly

with the parental

ODC

cDNA)

activity

some

in the stably

cells ODC

a property

cells or

but with

activity

trans-

remained

of transformed

cells in

mechanisms Because

DFMO,

Western

tyrosine

antibodies

of ODC

protein

blot analysis

stably transfected

was performed

in protein

had undergone

cell transformation

phosphorylation

lysates

cells. This revealed

the cells transfected is under

induced

tyrosine

using

derived a unique

augmented

by

antiphospho-

from

parental

and

band of 130 kilodal-

tyrosine

with ODC cDNA.

were

is reduced

phosphorylation

The identity

in

of this protein

investigation.

The authors

conclude

that ODC is necessary

tion and that the degree

of transformation

level of ODC,

implying

perhaps

prerequisites

for

might

the injection

involve

into colon

cancer

ated,

the introduction

and

immortalized

a certain

transformation.

upon

the

level and activity

are

Potential

future

of tumors, of ODC

an agent

such

cells into nude

the introduction if transformation into

colonic

as SV40

epithelial

serve if these cells can be transformed.

This work may function

into how ODC

cell proliferation

and oncogenesis,

cellular

pathways

regulating

pro-

activity

represent

areas of exciting

ODC

of ODC is acceler-

large T antigen

providing

new insights

experiments

of the stably transfected

cell lines to measure

with

for cell transforma-

is dependent

especially

cells to ob-

is important

in GI cancers.

and polyamine

in

in abnormal The

expression

and

investigation.

that increase with

a

mammalian

and then transhigh

in cancers

cells compared

cells: rate of growth,

of the promoters

of DNA

vectors

observed

effects

in in

this resulted

compared

in these

growth was variability

There

transcription.

Expression

to mimic

certain

vectors growth,

because

starvation,

in the

expression

cells.

and ODC

three

cDNA

led to accelerated

of the strength

a given

contains

to initiate

one can achieve

measure

in the present

that

In this fashion,

or and tu-

is to insert

vector

enhancer

promoters.

they

the ODC

fibroblast with

anchorage-independent

(when

mice to assay the formation

in malignant

of transformation

polymerase

may also contain

may repin the

activity

malignant

question

after

especially

polyamines

The promoter

RNA

be ele-

efficiently,

of

vector.

culture.

tons that ]

1986;255:G597-G601;

ODC

expression

the use of some eukaryotic

lated into protein.

and

et al. A general

in binding

may mucosa

of certain,

biomarkers

This fundamental

by Auvinen

activity

Res 1989;49:5884-5888) ODC

1987;60:

colonic

Furthermore,

may regulate

about

direct they

of cells.

in

(N Engl

ODC level and activity

phosphorylation

fied, membrane

condi-

is enhanced

polyps

hyperproliferation,

transformation and tyrosine

of

cells, a mouse

All three

the antisense

Furthermore,

to take up and the expression

in the expression

NIH3T3

vectors

mRNA

high even after serum Possible

post-

in a variety

Cancer

adenomatous ODC

cells.

ability

Furthermore,

containing

as a function

in ODC

by the cell type that

vary in their

DNA.

and

cells with

variability

to a given

transfected

ODC activity

prolifer-

is resistant

that

is determined

directions.

expression

the parental

of ODC

premalignant

or normal

these properties

the selection,

of after

marker

in the sense direction

phenotype,

The efficiency

based upon

vectors

in the stably

explored.

that ODC activity

mucosa

soft agar

cDNA.

are performed

can be transfected

and the anti-sense

any

perturbation

the cDNA

stably transfected

with ODC cDNA

in the

of the cDNA,

without

(involves

by the promoter

generally

expression

the different

by the level

of the ODC

Additionally,

gastric

Life Sci 1990;46:777-784).

morigenesis.

the

to be due to an

of regulation

and their corresponding

The authors

the trans-

overexpressing

cell types

is influenced

bound-

and serum

loop as determined

but also in benign

constitute

gastrin

is believed

of

before

mitogenic

has been observed

have shown

the cDNA

No. 4

direction

of techniques.

media)

added

alone

measurements

of a selectable

exogenously

sense

is at the level of ODC protein

expression

express

the ODC

phases

transition

of clones

involve

into a cell involves

by a variety

drug in the tissue culture

transformed

factor,

modification

cotransfection

controls

or opposite,

vector

DNA

the desired

Vol. 105,

is no transcription

or stable transfection

cell line that

1 amino acids

at the G2/M

means

hours)

and analysis

different

with certain

certain

(usually

ation,

Useful

of the

of exogenous

48-72

transcript

encoding to 46

phase

synthesis

feedback

involves

tions.

malignant

gene

(Genomics

(Mol Cell Biol 1988;9:5484-5490).

Deregulated human

frame

growth

This regulation

translationally

Of

of the ODC

corresponds

following

ODC

mRNA.

thesis or perhaps

ODC.

one ODC mRNA

at the Gl/S

increases

is by a negative

of polyamines.

is near

to hydroxyurea

and it also increases

ester, epidermal

Increased

transfection

is used as different

the gene for

mass of 51 kilodaltons.

peaks

synthesis

such as phorbol

2

retinoblas-

Furthermore, M2 subunit

which

11

The chromosomal

of ODC correlates

synthesis,

contains

two ODC gene

in that it is near N-m_yc,

The open reading

activity

the cell cycle. ODC ary. ODC

gene

contains

in neuroblastomas,

tissues express

molecular

The biological onset of DNA

ODC

genome

amplified

is 1383 nucleotides,

also

transient

reductase

Human

and

assays.

in the anti-sense,

such that there

of the cell membrane

in cells resistant

2.2 kilobases.

protein

RNA

by the use of

vector

inhibitor

of this gene as well as that

observed

1987;1:77-86).

cDNA

expression

irreversible

lung carcinomas.

ribonucletide

note,

DNA

of the cDNA

Transfection

of ODC is interesting

and small-cell

involved

tumorigenicity

fection

(dcSAM).

assisted

2 and 7, respectively.

(2p23ter)

a nuclear tomas,

study

been

notype,

are

2-difluoromethylornithine

The human

loci on chromosomes localization

whether

have

syn-

particular

(human,

and 12 exons.

activity

by spermine and spermine

S-adenosylmethionine

synthesis in

spermine

in spermidine

Sci 1990;15:153-158).

introns

resent

yields

groups

decarboxylated

Studies

Biochem

GASTROENTEROLOGY

alteration

levels of and then with

the

in phe-

Reply.

We would

like to thank

view of our findings future

experiments.

the ODC

and kind Noteworthy,

overexpressing

Dr. Rustgi

comments

our recent

studies

cells are also tumorigenic

tion of the pLTRpoly/ODC

transfected

NIH3T3

ously into nude mice gave rise to progressively all the cases, whereas

for his excellent

and outlining

the control

NIH3T3

re-

potential

indicate in vivo.

that Injec-

cells subcutane-

growing

tumors

cells were not tumori-

in